PROJECT SUMMARY Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer-enclosed particles with the biomolecular contents mirroring those of their parental cells. Since EVs are present in circulation at a relatively early stage of disease and persist across all disease stages, purification and characterization of tumor-derived EVs are expected to offer an opportunity for early cancer diagnosis. Hepatocellular carcinoma (HCC), the fourth most common cause of cancer-related deaths worldwide, is in dire need of diagnostic and prognostic biomarkers. Current clinical radiographic system and serum biomarkers (e.g., alpha-fetoprotein (AFP)) poorly discriminate early-stage HCC (where potentially curative therapies are available) from at-risk liver cirrhosis (where HCC surveillance is indicated). Moreover, sensitive biomarkers for HCC postoperative recurrence (where timely salvage treatment interventions can suppress disease progression) after curative-intent liver resection and liver transplantation remain a significant challenge for early-stage HCC. Therefore, exploiting the diagnostic potential of HCC EVs and EV cargo profiling for HCC early detection and postoperative recurrence holds great promise to significantly augment the ability of current diagnostic modalities. Conventional methods for isolating EVs, such as ultracentrifugation, filtration, and precipitation, are incapable of discriminating tumor-derived EVs from non-tumor-derived EVs. To address this unmet need, our team developed ?EV Click Chips? for HCC EV purification. The innovation of our devices includes i) the covalent chemistry-mediated EV capture/release couples click chemistry-mediated EV capture and disulfide cleavage- driven EV release, ii) an optimized multi-marker cocktail targeting HCC-associated surface markers was adopted to overcome the heterogeneity of HCC EVs; iii) the incorporation of densely packed silicon nanowire substrates (SiNWS) dramatically increases the device surface areas for contacting/interacting with EVs; and iv) the microfluidic chaotic mixer facilitates repeated physical contact between SiNWS and the flow-through EVs, further enhancing the performance of EV purification. The purified HCC EVs can be characterized by quantifying a panel of 10 well-validated HCC-specific mRNA markers by incorporating Droplet Digital PCR (ddPCR) technology. The proposed research will conduct: i) an exploratory development and optimization of EV Click Chips for HCC EV purification, and ii) clinical validations of EV Click Chips for HCC early detection and postoperative recurrence using patient blood samples. Our long-term goal is to develop a new HCC EV purification system (i.e., EV Click Chips) by synergistically integrating four very powerful approaches, including covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. The purified HCC EVs will readily allow for quantitative cargo profiling to augment current HCC diagnostic algorithms.